This application is based on application No 10-2001-78296 filed in the Korean Industrial Property Office on Dec. 11, 2001, the content of which is incorporated hereinto by reference.
(a) Field of the Invention
The present invention relates to recombinant adenoviruses expressing interleukin-18 proteins, and gene therapy using them. More particularly, the invention relates to a method of treating cancer cells by delivering the recombinant adenoviruses expressing interleukin-18 to cancer cells and inducing an immune response against tumor cells.
(b) Description of the Related Art
Cancer treatment is mostly carried out by surgical operation, irradiation, treatment using chemicals, methods using an immune promoting agent, etc. However, due to frequent recurrence and several side effects, more efficient and safer therapy has been required.
Due to the recent development of gene manipulation techniques, gene therapy using endogenous genes has been spotlighted in treating several congenital intractable diseases, as well as cancers. Gene therapy is a technique of treating diseases with proteins that are endogenously produced by injecting genes into cells and expressing them after the examination of molecular biological and biochemical causes of diseases. Gene therapy was attempted for the first time by the French Anderson group in America in September 1990, by introducing a gene into a patient suffering from combined immunodeficiency syndrome, and since then, more than 2,500 patients have been subjected to clinical trials.
For effective application of gene therapy to a cancer, the selection of therapeutic genes and vectors enabling selective and specific delivery to affected cells is important. Until now, the thymidine kinase of the herpes simplex virus, the cytosine deaminase of E.coli, p53, TRAIL (TNF-related apoptosis-inducing ligand), a gene for inhibiting angiogenesis, etc. have been used as therapeutic genes. As vectors, adenovirus, adeno-associated virus, retrovirus, liposome, and the like are used. However, those vectors are actually delivered to only a small portion of cancer cells when applied, and consequently, only a small portion of cancer cells express the therapeutic genes, and thus the efficiency of remedy is not that high (Mountain A, Trends in Biotechnology 18: 119-128, 2000).
If cytokines, immune response promoting factors, are used to remove tumors, they can induce a tumor-specific cellular immune response. In this case, even though therapeutic genes are introduced to a part of tumor cells, more effective remedy is possible because induction and systemic circulation of tumor-specific cytotoxic-T lymphocytes which causes the removal of tumor cells at distant site in which the therapeutic genes are not introduced.
Interleukin-18 (IL-18) is an immune response promoting factor, and it has also been known as interferon gamma inducing factor (IGIF). Interleukin-18 increases the cytotoxicity of T cells and NK cells, proliferates activated T cells, and stimulates Th1 cells to produce interleukin-2 and interferon-xcex3 (Dinarello C. A. et al., Methods 63:658-644, 1998). Also, it inhibits the synthesis of IgE by inducing interferon gamma from B cells, increases the production of granulocyte-macrophage colony stimulating factor (hereinafter referred to as xe2x80x98GM-CSFxe2x80x99), and promotes the production of interleukin-2. In addition, it inhibits the production of interleukin-10, an immune suppressing cytokine.
Interleukin-18 is synthesized as a precursor in a biologically inactive state, and then cleaved with interleukin-1xcex2-converting enzyme (ICE, caspase-1), a cysteine protease present in the cells, to be converted into an active form. The precursor or activated mature interleukin-18 is digested with caspase-3 (CPP32) and is thus inactivated.
The gene of interleukin-18 has been isolated from humans (GenBank accession number E17135) and mice (GenBank accession number E17139) and recently, anticancer efficiency tests wherein interleukin-18 proteins are administered directly into mice have been attempted, and it has been reported that they exhibit effects to some degree. It is believed that interferon gamma produced from T cells or activated NK cells have a key role in those effects (Micallef et al. Cancer Research 57, 4557-4563, 1997). Accordingly, if in the invention, interleukin-18 is injected into cancer cells as a DNA and not a protein, by adenoviral vectors enabling its overexpression it is expected that more effective anticancer treatment can be achieved.
It is an object of the invention to provide an interleukin-18 protein mutant that has the interferon gamma inducing ability and is not inactivated by caspase-3.
It is a further object of the invention to provide a gene encoding an interleukin-18 protein mutant that has the interferon gamma inducing-ability and is not inactivated by caspase-3.
It is a further object of the invention to provide a method enabling an interleukin-18 protein to be extracellularly expressed.
It is a further object of the invention to provide a method enabling an interleukin-18 protein to be expressed in tumor cells.
It is a further object of the invention to provide a vector capable of expressing an interleukin-18 protein and a mutant interleukin-18 protein.
It is a further object of the invention to provide a recombinant virus capable of expressing an interleukin-18 protein and a mutant interleukin-18 protein.
It is a further object of the invention to provide a method of treating tumor cells using a recombinant adenovirus expressing interleukin-18.
To accomplish the aforementioned objects, the invention provides an interleukin-18 protein wherein a cleavage site for caspase-3 is substituted by an amino acid selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
Also, the invention provides an interleukin-18 gene comprising a nucleotide sequence encoding the interleukin-18 protein.
Also, the invention provides a recombinant adenovirus comprising (a) a promoter operably linked to an interleukin-18 gene so that it can be expressed, (b) the interleukin-18 gene, and (c) a polyadenylation signal sequence.
Also, the invention provides a method for inhibiting the proliferation of tumor cells by injecting the recombinant adenovirus into cells and expressing the recombinant interleukin-18 protein.
Also, the invention provides a method of expressing an interleukin-18 protein capable of inhibiting the proliferation of tumor cells and enhancing an immune response, and of secreting it extracellularly.